The studies in this grant are focussed on the potential functions of tropomodulin, first in the length regulation of actin filaments in cells such as the red blood cell and muscle and second, in the regulation of actin assembly of actin based motility in non-muscle cells. Tropomodulin is a novel 40.6kD protein with binding sites for actin and tropomyosin, a coiled coil that binds alongside the actin filament. Very recently, we showed in collaboration with V.M.Fowler that tropomodulin has two different functions. First, in the presence of tropomyosin it acts as a tight cap at the pointed end of actin filaments. This function is probably very important in stabilizing the pointed ends of actin filaments whose length is tightly regulated as in the membrane skeleton of red blood cells. Second, alone, tropomodulin is a leaky cap which increases the pointed end critical concentration which may play an important role in the regulation of actin assembly of actin based motility in non-muscle cells. In both of these functions tropomodulin displays very unusual properties not observed with any other capping proteins. As part of our proposed studies on the mechanism of action of the tight capping, evaluating the relative roles of tropomodulin and of tropomyosin, we plan to determine the reason why, unlike any other capping protein, tropomodulin + tropomyosin do not cap all of the actin filaments equally. For instance, to cite just one possible cause, it may be that very short actin filaments, like those in the red blood cell, are excluded from capping because, without the participation of additional proteins, more than one tropomyosin molecule per act in strand is required for tight capping. In view of the unique sensitivity of the extent of capping to the conditions of the assembly of the tropomodulin- tropomyosin-actin filaments we intend to investigate a similar sensitivity in the behavior of tropomyosin-actin filaments in the absence of tropomodulin. For instance, under some conditions of copolymerization of actin with tropomyosin, we found a severe inhibition of elongation at the barbed filament end which might be important for the stabilization of the barbed ends of the actin filaments in the red blood cell where no barbed-end -capping protein has yet been identified. As part of our studies on the mechanism by which tropomodulin in the absence of tropomyosin increases the pointed end critical concentration and acts as a leaky cap we will pay special attention to the involvement of ATP hydrolysis in this process.